High-Level Tissue-Specific Expression of Functional Human Factor VIII in Mice

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Abstract
Hemophilia A results from subnormal levels of blood coagulation factor VIII (FVIII) and is an attractive target for gene therapy. However, progress has been impeded by features of FVIII biology such as low mRNA accumulation and the instability of the protein. We have shown previously that a FVIII adenoviral vector, Av1ALH81, allowed high-level expression of human FVIII in mice sustained for several weeks. Here, we have generated a second FVIII adenoviral vector, Av1ALAPH81, in which an intron was introduced into the FVIII expression cassette. Administration of Av1ALAPH81 to mice resulted in significantly increased FVIII plasma levels, 1,046 ± 163 ng/ml compared to 307 ± 93 ng/ml of FVIII detected in mice that received Av1ALH81. Normal FVIII levels in humans are 100–200 ng/ml and therapeutic levels are as low as 10 ng/ml. Therapeutic levels are defined as the amount of FVIII necessary to convert severe hemophilia to a moderate or mild hemophiliac condition. The increased potency of the second FVIII adenoviral vector allowed the administration of significantly lower, less toxic vector doses, while retaining the potential for high FVIII expression. Furthermore, we demonstrate that adenoviral-mediated expression of human FVIII can be limited to the liver by inclusion of a liver-specific promoter, thereby achieving the first step in regulated expression of human FVIII in vivo. Factor VIII (FVIII) deficiency results in hemophilia A, a common hereditary bleeding disorder. We have demonstrated previously the feasibility of direct in vivo adenoviral transfer and expression of human FVIII. However, the vector dose necessary to achieve therapeutic FVIII blood levels was hepatotoxic. In this work, we report the generation of a second, more potent FVIII adenoviral vector that allows high-level FVIII expression with administration of lower, less toxic vector doses. In addition, the inclusion of a liver-specific promoter into the adenoviral vector limited expression of human FVIII to the liver. The generation of potent adenoviral vectors that, at low doses, mediate high-level, liver-specific expression of human FVIII in vivo provides a greater margin of safety for potential gene therapy-based treatments of hemophilia A.